Pruritus is a major symptom of dermatologic and internal conditions. It can be difficult to treat, as few specific inhibitors of itch are available and the mechanism that triggers the sensation of itch is not clear. Although most experimental studies of itch use histamine as the pruritic stimulus, most cases of clinical pruritus are considered to be histamine independent. The discovery of natural compounds that evoke itch without releasing histamine might facilitate the search for endogenous mediators and receptors distinct from histamine. Spicules of the plant Mucuna pruriens (cowhage) when lodged in the epidermis produce moderate to severe itching that is independent of histamine. We have determined that the active compound in cowhage is mucunain, a cysteine protease. We have also determined that mucunain is a ligand for human protease-activated receptors 2 and 4. We hypothesized that human cysteine proteases might share homology with mucunain, activate these same receptors, and function as endogenous mediators of pruritus. We provide data that this is the case with certain cathepsins, notably Cathepsin S. Cathepsins have been implicated in many processes but have not previously been considered signaling molecules. The experiments proposed in Aim 1 are designed to decipher the mechanism by which cysteine proteases activate their cognate receptors. Experiments proposed in Aim 2 with receptor knockout mice will determine if such receptors are indeed necessary for the nociceptive effects of cathepsin S. The promoter region of cathepsin S contains STAT binding sites leading us to hypothesize that the critical pruritus-associated cytokine IL-31 activates its receptor leading to the induction of cathepsin S expression. This hypothesis will be tested in Aim 3. The hypotheses and experiments presented here link together three molecules that have previously been associated independently with itch and inflammation: PARs, cathepsins and IL-31. PUBLIC HEALTH RELEVANCE: Itching is a major symptom of dermatologic and many internal conditions and is difficult to treat. The goal of this project is to examine how certain proteins, called cysteine proteases, not previously associated with itching, appear to turn on specific receptors and cause itching. The results of this project may lead to the development of new drugs to treat itch.